Characterization of the Gene Expression Profile of Human Bocavirus

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Virology 403 (2010) 145–154

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Virology

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Characterization of the gene expression proﬁle of human bocavirus

Aaron Yun Chen a,1, Fang Cheng a,1, Sai Lou a,b, Yong Luo a, Zhengwen Liu b, Eric Delwart c, David Pintel d, Jianming Qiu a,⁎

a Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS, USA b Department of Infectious Diseases, The First Afﬁliated Hospital, Xi'an Jiaotong University, Xi'an, China c Blood Systems Research Institute, San Francisco, CA, USA d Life Sciences Center, University of Missouri-Columbia, Columbia, MO, USA

article info abstract

Article history: We have generated a quantitative transcription profile of human bocavirus type 1 (HBoV1) by transfecting a Received 16 February 2010 nearly full-length clone in human lung epithelial A549 cells as well as in a replication competent system in Returned to author for revision 9 April 2010 293 cells. The overall transcription profile of HBoV1 is similar to that of two other members of genus Accepted 15 April 2010 Bocavirus, minute virus of canines and bovine parvovirus 1. In particular, a spliced NS1-transcript that was Available online 10 May 2010 not recognized previously expressed the large non-structural protein NS1 at approximately 100 kDa; and the fi Keywords: NP1-encoding transcripts were expressed abundantly. In addition, the protein expression pro le of human Human bocavirus bocavirus type 2 (HBoV2) was examined in parallel by transfection of a nearly full-length clone in A549 cells, Gene expression which is similar to that of HBoV1. Moreover, our results showed that, unlike human parvovirus B19 infection, Protein expression of the HBoV1 proteins only does not induce cell cycle arrest and apoptosis of A549 cells. © 2010 Elsevier Inc. All rights reserved.

Introduction However, HBoV1 acute infection with high viral loads in respiratory samples (N10,000 copies/ml) and increased IgG or IgM detection have Human Bocavirus (HBoV) is one of the recently identified been frequently associated with acute respiratory illnesses (Allander respiratory viruses, and tentatively is classified in the genus Bocavirus et al., 2005; Kantola et al., 2008), which indicates an apparent within the subfamily of Parvovirinae of the Parvoviridae family etiological link to respiratory illnesses. In addition to respiratory (Cotmore and Tattersall, 2005). Other members in the Bocavirus illnesses, HBoV1 is associated with gastroenteritic diseases (Lee et al., genus are bovine parvovirus type 1 (BPV1) (Chen et al., 1986), minute 2007; Arnold et al., 2006; Lau et al., 2007; Vicente et al., 2007; virus of canines (MVC) (Schwartz et al., 2002). Different species of Albuquerque et al., 2007), a characteristic that shares with two closely HBoV have been identified in humans including the prototype HBoV related animal bocaviruses. The largest genome of HBoV1 that has in respiratory samples as well as HBoV2 and HBoV3 in feces (Kapoor been sequenced is 5299 nts, which lacks both termini; therefore, it is et al., 2009; Arthur et al., 2009). not infectious. To date, both termini of the HBoV genome have not HBoV1 was initially identified from nasopharyngeal aspirates of been sequenced; therefore, an HBoV1 infectious DNA clone has not patients with lower respiratory infections (Allander et al., 2005). The been described. Recently, new species of human Bocavirus, HBoV2 and HBoV1 genome has been frequently detected worldwide, ranging HBoV3, were identified in human stool specimens. HBoV2 has a from 2% to 19% in respiratory specimens from children under 2 years genomic organization identical to that of HBoV1, but the HBoV2 NS1, old with acute respiratory illnesses (Allander et al., 2005; Arden et al., NP1, and VP1 proteins have only 78%, 67%, and 80% identity to those of 2006; Arnold et al., 2006; Bastien et al., 2006; Choi et al., 2006; HBoV1, respectively. Further studies are necessary, however, to Foulongne et al., 2006; Ma et al., 2006; Sloots et al., 2006; Weissbrich identify potential associations of HBoV2 and HBoV3 with clinical et al., 2006; Lin et al., 2007, 2008). HBoV1 is associated with acute symptoms or disease (Kapoor et al., 2009; Arthur et al., 2009). expiratory wheezing and pneumonia (Allander et al., 2007; Kahn, A cell culture system of HBoV1 infection has been recently 2008; Schildgen et al., 2008), and is commonly detected in association established (Dijkman et al., 2009); however, it is inefficient in that with other respiratory viruses (Kahn, 2008; Schildgen et al., 2008). HBoV1 transcripts were only detected by reverse-transcription (RT)- PCR. Six transcripts of HBoV1 were identified from HBoV1-infected differentiated human airway epithelial cells (Dijkman et al., 2009). ⁎ Corresponding author. Department of Microbiology, Molecular Genetics and The abundance of these transcripts and their coding capabilities are Immunology, University of Kansas Medical Center, Mail Stop 3029, 3901 Rainbow not yet understood. Two non-structural proteins NS1 and NP1 were Blvd., Kansas City, KS 66160, USA. Fax: +1 913 588 7295. E-mail address: [email protected] (J. Qiu). predicted, but the NS1 seems to lack the C-terminus compared to the 1 These authors contributed equally. full length NS1 of BPV1 and MVC (Qiu et al., 2007; Sun et al., 2009).

The full length sequence of infectious MVC DNA (Genbank accession infection system (Dijkman et al., 2009), with the exception of an extra no.: FJ214110) shows 52.6% identity to HBoV1, while the NS1, NP1 splice site (A1-1) in the first intron. This A1-1 splice site is present in and VP1 proteins of MVC are 38.5%, 39.9% and 43.7% identical to those BPV1 RNA during BPV1 infection (Qiu et al., 2007), but not in MVC RNA of HBoV1, respectively (Sun et al., 2009). We have previously from MVC infection (Sun et al., 2009). determined the transcription profiles of the BPV1 and MVC during To determine the relative abundance of each HBoV1 mRNA, we infection (Qiu et al., 2007; Sun et al., 2009). In BPV1, the left ORF used seven anti-sense HBoV1 probes to protect individual HBoV1 encodes the non-structural protein NS1, at approximately 100 kDa, mRNA. A schematic diagram of the seven anti-sense HBoV1 probes and proteins at relatively small sizes are proposed as NS2 (Qiu et al., with their putative protected bands and nucleotide numbers (nt) is 2007; Lederman et al., 1987). The mid-ORF is thought to encode the shown in Fig. 1A. BPV1 abundant non-structural protein NP1 at 28 kDa, and the right ORF contains the coding sequences for the overlapping capsid protein Probe PD1 genes VP1 and VP2 (Qiu et al., 2007; Lederman et al., 1987). Both the NS1 and NP1 of MVC are important in DNA replication of MVC (Sun Probe PD1, spanning the putative promoter (P3) and the first et al., 2009). The NP1 of HBoV1 and BPV1 can supplement the lack of donor site (D1), protected bands of 133 and 55 nts. These bands function of the MVC NP1 in replication of an NP1 knock-out MVC mapped the RNA initiation site at nt 187 and the first splice donor site infectious clone to some degree (Sun et al., 2009); however, the NP1 (D1) at nt 242. Similar to that of BPV1 and MVC, the first exon of HBoV proteins of the bocaviruses share no similarity to any proteins of the is short, containing only 55 nts (Fig. 1B, lane 2). Approximately 75% of other parvoviruses. Detection of HBoV proteins either during infection HBoV1 RNAs were spliced at the D1 donor site. Multiple bands or in transfection has not been reported. centralized at nt 55 were considered as spliced RNA (Qiu et al., 2002). In this study, we generated a comprehensive transcription profile of HBoV1 by transfecting a replicative chimeric HBoV1 genome in 293 Probe PA1-1 cells and a non-replicative genome in A549 cells. We studied the expression profiles of both the structural and non-structural proteins Probe PA1-1, spanning the first acceptor site of the first intron (A1- of HBoV in detail. Transcripts encoding the left ORF (NS1) are either 1), protected bands of 182 and 120 nts (Fig. 1B, lane 3). The 120-nt spliced or unspliced at a small intron that lies in the middle of the band mapped the first 3′ splice acceptor site at nt 920, which is similar genome. Thus, the spliced transcripts are able to encode a large to the location of the acceptor site in BPV1 RNA. In addition, as seen in nonstructural protein NS1 at approximately 100kDa, which is BPV1, only a small portion of RNAs, less than 10% of spliced RNA at the comparable to the NS1 of MVC and BPV1; while the unspliced D1 donor site, were spliced at the A1-1 acceptor site (Qiu et al., 2007). transcripts encode a relatively small nonstructural protein NS1-70 at approximately 70 kDa. Probe PA1-2 and PA1-2/D2

Results Probe PA1-2, spanning the second acceptor of the first intron (A1-2), protected bands of 162 and 117 nts (Fig. 1C, lane 2). Probe PA1-2/D2, Determine the relative abundance of HBoV1 transcripts by RNase spanning the second acceptor of the first intron (A1-2) and the donor protection assay (RPA) site of the small intron (D2), protected bands of 225, 122 and 168 nts (Fig. 1C, lane 3). These bands protected from both probes confirmed the A transcription map of the HBoV1 has been reported, which was location of the A1-2 acceptor and the D2 donor at nt 2043 and nt 2165, obtained from HBoV1 RNA isolated from HBoV1-infected differentiated respectively. Similar to BPV1 and MVC, the majority of HBoV1 RNAs human airway epithelial cells (Dijkman et al., 2009). In that report, the protected by probe PA1-2/D2 were spliced at both sites, resulting in the HBoV1 transcripts were identified by reverse transcription (RT)-PCR; abundant band present at 122 nts (approximately 90%). Only approx- therefore, the relative abundance of the individual transcripts could not imately 5% of RNAs in this region were unspliced at the A1-2 acceptor be determined. In other parvovirus systems, little difference has been site, which resulted in the band at 168 nts (spliced at D2) and the band seen between viral infection and plasmid transfection (Qiu et al., 2002, at 225 nts (unspliced at D2). Interestingly, this probe did not detect 2005, 2007; Sun et al., 2009). Therefore, we decided to examine the RNAs that were spliced at A1-2 and remained unspliced at the D2 site HBoV1 transcription profile in detail by transfection. To this end, we (which would have generated a band at 181 nts). constructed an HBoV1 plasmid (pHBoV1) containing the full HBoV1 coding sequence (nt 1–5299) by amplifying a nearly full-length genome Probe A2/D3 from an HBoV1-positive nasopharyngeal aspirate sample. The sequence of this clone was deposited in Genbank (access no.: GQ925675). We Probe PA2/D3, which spans the A2 acceptor and the D3 donor sites, have found previously that the status of replication alters the protected bands at 241, 123, 192 and 167 nts. These bands confirmed transcription profile of human parvovirus B19 (B19V) (Guan et al., the usage of the A2 site at nt 2235 and the D3 site at nt 2358 (Fig. 1D, lane 2008). To observe the transcriptional profile in a replication-competent 2). Approximately half of the RNAs that spliced the small intron (D2–A2) system, we inserted the HBoV1 genome into adeno-associated virus were not further spliced at the third intron (D3–A3), which resulted in type 5 (AAV5) inverted terminal repeats (ITRs) so that it could be the 192-nt band; another half were spliced at D3, which resulted in the replicated while AAV5 Rep78 and necessary Ad5 genes were provided 123-nt band. These spliced mRNAs at 123 nts (spliced at both A2 and D3, (Guan et al., 2008) in 293 cells (data not shown). Fig. 1D, lane 2) represent capsid protein-encoding mRNAs, which We used mRNAs isolated from two sources for transcript mapping: account for one third of the RNAs protected by probe A2/D3. Only a those from A549 cells transfected with pHBoV1, and those from 293 minor portion of RNAs, at a size of 167 nts, were unspliced at the A2 site cells transfected with p5TRHBoV1/pHIVAV5Rep78/phelper. RT-PCR and spliced at the D3 site (Fig. 1D, lane 2), which was not seen in RNAs and 5′/3′ RACE were performed as previously described (Qiu et al., from transfected A549 cells (data not shown); therefore, we did not 2002, 2005, 2007; Sun et al., 2009), and gel purified PCR fragments were include this transcript in the final transcription profile. further sequenced (data not shown). The RNA landmarks determined by transfection of p5TRHBoV1 in 293 cells, which were the same as Probe PA3 those determined by transfection of pHBoV1 in A549 cells, are depicted and diagramed to scale in Fig. 1A. These landmarks basically confirmed Probe PA3, which spans the A3 acceptor site, protected two bands the transcription units as identified in the previously reported virus at 159 and 107 nts. Thus the A3 acceptor site was mapped to nt 2995 A.Y. Chen et al. / Virology 403 (2010) 145–154 147

Fig. 1. Transcription mapping of HBoV1 RNA by RPA. (A) Schematic diagram of the HBoV1 genome and the probes used for RPA. The landmarks of transcription: the promoter (P3), the splice donor sites (D1, D2, and D3), the acceptor sites (A1-1, A1-2, A2 and A3), the internal polyadenylation signal (pA)p and the distal polyadenylation site (pA)d, which were identiﬁed by RT-PCR and RACE, are shown. The RPA probes PD1 (nt 161 to 320), PA1-1 (nt 854 to 1040), PA1-2 (nt 1998–2160), PA1-2/D2 (1998–2223), PA2/D3 (nt 2191–2432), PA3 (nt 2961–3120) and p(pA)p (nt 3291–3440) are shown, along with the designated bands they are expected to protect and their predicted sizes. (B, C, D and E) Mapping of the HBoV1 transcription map by RPA. Ten µg of total RNA isolated 2 days from p5TRHBoV1-transfected 293 cells was protected by the above described probes, in individual reactions as indicated. Lane 1, 32P-labeled RNA markers (Qiu et al., 2002), with sizes indicated to the left. The origins of the protected bands in the lanes are indicated. Spl, spliced RNAs; Unspl, unspliced RNAs; RT, RNAs read through the (pA)p site; (pA)p, polyadenylated RNAs at (pA)p.

(Fig. 1E, lane 2). The ratio of unspliced to spliced RNA across this ratio of RNAs that read-through vs. those polyadenylated at (pA)p was region was approximately 1:1. approximately 1:1. The AAUAAA at nt 3232 was not used (data not shown). Probe P(pA)p Northern blot analysis of HBoV1 RNA Probe P(pA)p was used to map the internal poly A signal (pA)p (Fig. 1E, lane 3). The band at 108 nts mapped the cleavage site at 3406, From the results obtained from RT-PCR and RPA, we obtained a consistent with the site identiﬁed by 3′RACE (data not shown). The preliminary transcription map of HBoV1 expression by transfection 148 A.Y. Chen et al. / Virology 403 (2010) 145–154

(Fig. 2B). We next sought to determine the abundance of these HBoV1 that all the R5, R6 and R7 are putative mRNAs to encode capsid transcripts by Northern blot analysis using three probes that are proteins (Fig. 3A). schematically diagramed at the bottom of Fig. 2B. The NS probe should detect all the species of HBoV1 RNAs because Hybridization of HBoV1 RNAs, either from 293 cells (replication- they are generated from a single promoter and share the same first competent) or A549 cells, with the Cap probe revealed HBoV1 exon (only 60 nts). Similar to MVC and BPV1 RNAs, the first exon is transcripts that were polyadenylated at the (pA)d site (Fig. 2A, lanes 2 short, and the NS probe hybridized to these first-exon-containing and 3). These transcripts were the 4.8 kb R2M mRNA and the 4.9 kb RNAs poorly; therefore, hybridization of HBoV1 RNAs with the NS R2m mRNA, which putatively encode NS1, the 3.0 kb R4 mRNA— probe only revealed two abundant RNA species, i.e. R1M at 3.1 kb and putatively encoding NP1 as well as the 2.4 kb R5 mRNA—putatively R2M at 4.8 kb (Fig. 2, lanes 4 and 5), both of which could encode NS1. encoding capsid protein VP1 and VP2. The R6 mRNA that was detected R1m RNA at 3.2 kb was not clearly detected in either HBoV1 RNA in HBoV1 RNA from infected cells (Dijkman et al., 2009) was predicted sample. to hybridize with the Cap probe at a size of 2.3 kb. The R7 mRNA, Northern analysis with the whole HBoV1 genomic probe (NSCap) which is similar to that encoding MVC and BPV1 capsid proteins, was revealed the overall HBoV1-generated RNA profiles from transfection detected by RT-PCR in RNAs isolated from both transfection systems of both replicative and non-replicative plasmids (Fig. 2A, lanes 6 and (data not shown); it was predicted to hybridize with the Cap probe at 7, respectively). Eight major species of RNAs were detected, and these a size of 2.1 kb (Fig. 2A, lanes 2 and 3). Since the Northern blot did not were consistent in size with RNAs predicted to encode NS1 (R1M and resolve the bands at sizes of 2.1, 2.2 and 2.4 kb very well, we assume R2M mRNA at 3.1 and 4.8 kb, respectively), NS1-70 (R2m mRNA at

Fig. 2. Transcription profile of HBoV1 RNA. (A) Northern blot analysis of HBoV1 RNA. Total RNAs were extracted from p5TRHBoV1-transfected 293 cells or pHBoV1-transfected A549 cells, mRNAs purified from 5 or 20 µg of the total RNAs were loaded in the lane as indicated for Northern blot. The blots were hybridized to NS, Cap and NSCap probes, respectively, which are diagramed at the bottom of panel B with their specific locations. The size of each detected bands is shown to the right of each lane. The identities of each band are illustrated in panel B. RNA maker ladder (Ambion) is shown in lane 1. The asterisk indicates a band at approximately 1.8 kb, which was detected by all the three probes. (B) Putative transcription map of HBoV1. The genome of HBoV1 is shown to scale with transcription landmarks, including the P3 promoter, splice donor (D1, D2 and D3) and acceptor (A1-1, A1- 2, A2 and A3) sites, the internal polyadenylation site [(pA)p], and the distal polyadenylation site [(pA)d]. All of the RNA species are proposed from results of RT-PCR and RPA and shown with their respective sizes (minus a poly A tail of approximately 150 nts). The putative ORFs are diagramed with their predicated size in kDa of translated proteins. The cleavage sites are shown by arrows with respective nucleotide numbers. HA-tagged ORFs are indicated by a “diamond” symbol. A.Y. Chen et al. / Virology 403 (2010) 145–154 149

Fig. 3. Genetic map of HBoV1 and alignment of Bocavirus NS1 C-terminus. (A) A summarized genetic map of HBoV1 is shown with transcription landmarks in panel A. Six major species of HBoV1 transcripts that were conﬁrmed by Northern blot analysis are shown with their relative abundance and sizes (minus a poly A tail of approximately 150 nts). Proteins detected by transfection are indicated. (B) Comparison of the NS1 C-termini of HBoV1, BPV and MVC. The C-terminus aa 639–781 of HBoV NS1, aa 662–774 of MVC NS1, and aa 733–860 BPV NS1 were chosen to align using ClustalW2 (Larkin et al., 2007). Identical amino acids are shown as a “star” symbol, while homology amino acids shown as two dots under the amino acid sequences of the C-termini. Conserved motifs are underlined.

4.9 kb), NP1 (R3 and R4 mRNAs at 1.3 and 3.0 kb, respectively), and always detected by all the three probes but not in RNAs extracted VP1/2 (R5, R6 and R7 mRNA at 2.4, 2.2 and 2.1 kb, respectively). R3 from mock transfected cells (data not show). The nature of this 1.8 kb and R4 mRNAs, both of which could encode NP1, are the most band is currently unknown; however, a similar band was also abundant RNAs and account for approximately 50% of total HBoV1 detected in Northern blot analysis of MVC RNA (Sun et al., 2009). RNAs (Fig. 2A, lanes 6 and 7, 1.3 and 3.0 kb). The overall abundance of Summarizing results from RPA and Northern blot analysis, we the R5, R6 and R7 capsid protein-encoding mRNAs constitute generated a genetic map of HBoV1, which is shown in Fig. 3A. Eight approximately one third of total HBoV1 RNAs (Fig. 3A). major species of HBoV1 mRNAs are shown with their respective sizes, Although the A1-1 acceptor was shown by the RPA using a probe abundance in percentage and proteins to encode. This revised map is spanning the acceptor, the R8 and R9 mRNAs, which may use very similar to that of MVC (Sun et al., 2009). alternative polyadenylation, were not clearly hybridized as bands at 1.8 and 3.5 kb. Similar mRNAs were obvious on the Northern blot of Expression of HBoV proteins BPV-1 RNA but not on the blot with MVC RNA (Sun et al., 2009). Interestingly, in HBoV1 RNAs extracted from infected cells, such Next, we used the transcription map as a guide to profile HBoV1 spliced R8 and R9 mRNAs were not detected by RT-PCR (Dijkman non-structural proteins expressed by transfection. First, we tagged an et al., 2009). However, since tissue-specific splicing has been observed HA-tag to the N-terminus of the NS1 ORF in the full length clone of in parvovirus MVM pre-mRNA processing (Choi et al., 2005), the pHBoV1 to make HA-NS1-expressing plasmid pHBoV1(HA-NS1), and significance of the R8 and R9 mRNAs awaits further investigation. A an HA-tag to the C-terminus of the NP1 ORF in pHBoV1 to make NP1- band at approximately 1.8 kb, indicated by an asterisk in Fig. 2A, was HA-expressing plasmid pHBoV1(NP1-HA). Transfection of pHBoV1 150 A.Y. Chen et al. / Virology 403 (2010) 145–154

(HA-NS1) revealed two bands at approximately 100 and 70 kDa antiserum revealed three major bands, at approximately 100, 66 and (Fig. 4A), confirming that R1M and R2M mRNAs encode the large NS1 34 kDa, respectively (Fig. 4B, lane 2). The sizes of these bands were protein, and R2m mRNA encodes the small NS1-70 protein. confirmed by resolving the NS1 proteins on SDS-6% and -12% PAGE Transfecting pHBoV1(NP1-HA) confirmed the expression of NP1 at gels, respectively (Fig. 4B). approximately 26 kDa from R3 and R4 mRNA (Fig. 4A, lane 3). This antiserum did not react with the NS1-70 (Fig. 4B, lane 1). Thus Since a large size of the NS1 (at ∼100 kDa) was detected in cells we have determined that the HBoV1 NS1 was expressed as a full transfected with pHBoV1, we aligned the C-terminus of the NS1 length protein of 100 kDa, and small proteins at 66 and 34 kDa, proteins of HBoV1, BPV and MVC, as shown in Fig. 3B, using ClustalW2 respectively. The mechanism by which the C-terminal 66-kDa (Larkin et al., 2007). The result showed that there are two conserved (NS1*66) and 34-kDa (NS1*34) proteins were generated is currently motifs, 740WGERLGLI747 and 757PIVLXCFE764, present at the C- unknown. Interestingly, similar small NS1 proteins were detected in terminus of the Bocavirus (Fig. 3B). We were not able to identify pHBoV2-transfected cells (Fig. 4B, lane 3). The intact HBoV1 and similar motifs in the NCBI BLAST database (http://www.ncbi.nlm.nih. HBoV2 NS1 were detected at approximately 100 kDa rather than at gov/Structure/cdd/wrpsb.cgi), suggesting that they are highly con- theputativesizeat85kDa(Sun et al., 2009), indicating a served motifs among members of the genus Bocavirus. However, the modification, perhaps phosphorylation, of both NS1. The sizes of search engine failed to identify any known domain with expected HBoV2 NS1 and NS1*66 were further confirmed by resolving the NS1 value lower than 1. Nevertheless, the potential role of these highly proteins on an SDS-6%PAGE gel (Fig. 4B, 6%PAGE, lane 2). As expected, conserved motifs of Bocavirus NS1 C-terminus warrants further study. the anti-HBoV1 NP1 antiserum detected both HBoV1 and HBoV2 NP1 To further confirm results obtained from the two HA-tagged at approximately 26 kDa in transfected cells (Fig. 4C, lanes 1 and 2), constructs and the conserved C-terminus of the NS1, we raised suggesting they are not modified unlike the HBoV1 and 2 NS1 antiserum against the C-terminus of NS1, which shares a portion of proteins. coding sequences with NP1 but in different frames, and antiserum We chose GST-fused HBoV1 VP1 peptide of aa 369–475 to generate against the whole NP1, respectively. Western blot analysis of cell antiserum. In this region, two identical domains, aa 380–400/aa 376– lysate from pHBoV1-transfected cells with anti-NS1 (C-terminus) 396 and aa 422–440/aa 418–436, are present both in HBoV1 and

Fig. 4. Expression of HBoV proteins. (A) Western blot analysis of HBoV1 protein by transfection of HA-tagged constructs. A549 cells were transfected with HA-tagged constructs as indicated. Two days posttransfection, cells were lysed for Western blot using a monoclonal antibody against HA tag (HA-7, Sigma). (B, C and D) Western blot analysis of HBoV1 and HBoV2 proteins by transfection. A549 cells were transfected with the nearly full-length clones of pHBoV1 and pHBoV2 as indicated. Two days later, cells were lysed for Western blot using anti-HBoV1NS1 (B), anti-HBoV1NP1 (C), and anti-HBoV1VP1/2, respectively. The identities of detected proteins are shown to the left on the blot. Small bands of the NS1 at 66 and 34 kDa that retain the C-terminus are shown as NS1*66 and NS1*34, respectively. pBluescript SK(+)-transfected cells were used as control (Ctrl). A.Y. Chen et al. / Virology 403 (2010) 145–154 151

HBoV2 VP1. By using the anti-VP1 antiserum, we confirmed the viral infection is important in the virus life cycle, particularly for viral putative VP1 and VP2 of both HBoV1 and HBoV2 at approximately 72 DNA replication and virus egress. Therefore, we decided to examine and 60 kDa, respectively (Fig. 4D). The anti-VP1/2 antiserum cross the apoptotic cell death and cell cycle regulation induced by HBoV1 reacted with a cellular protein at a size of approximately 90 kDa as viral proteins. We transfected the HBoV1 non-replicative construct indicated by an arrow (Fig. 4D). These results suggest a similar pHBoV1 into A549 cells. Transfected cells were analyzed for cell cycle expression of NS1, NP1 and VP1/2 between the two species of HBoV. and apoptosis by DAPI and FLICA staining, respectively. Both NS1- Moreover, the conserved expression of the small NS1*66 in cells expressing and non-expressing cell populations were gated and transfected with both pHBoV1 and pHBoV2 suggest that it must be plotted for comparison (Fig. 6). Expression of the NS1, NS1*66, important in Bocavirus infection. NS1*34, NS1-70, NP1 and VP1/VP2 of HBoV1 in transfected cells were Proteins at sizes of 47 kDa, potentially expressed from putative R8 detected by Western blot (Fig. 4). However, transfection of pHBoV1 and R9 mRNAs, were not detected with anti-HA, anti-NS1 or anti-NP1 did not induce an obvious cell cycle change and apoptotic cell death in antiserum, further supporting that mRNA spliced at A1-1 may not be NS1 expressing cells (NS+) compared to those in NS1(−) cell significant. In addition, protein bands at 17.5 and 8.8 kDa, which population [Fig. 6, compare numbers in NS+ with NS1(−)]. Active include putative UP1 and UP2 ORFs, respectively, from the previously caspase is a hallmark of apoptotic cell death (Degterev et al., 2003). identified map of HBoV1 generated from HBoV1-infected cells Thus, our results suggest that expression of both HBoV1 non- (Fig. 2B) (Dijkman et al., 2009), were not detected in pHBoV1 and structural and structural proteins does not induce an apparent 2-transfected cells by either anti-NS1 (Fig. 4B, lanes 2 and 3) or anti- apoptosis or a perturbation of cell cycle progression in transfected HA (Fig. 4A, lanes 2 and 3). cells. A genetic map of HBoV1 summarizing the results of the experiments so far described is shown in Fig. 3A. Collectively, R1M Discussion and R2M mRNAs encode the large NS1 protein at a size of approximately 100 kDa. The HBoV1 NS1 protein apparently was HBoV RNA profile modified, perhaps phosphorylated, since its coding capability is only 86 kDa. Interestingly, at least two small stable NS1 proteins, NS1*66 In this paper, we report a detailed characterization of the and NS1*34 at 66 and 34 kDa, respectively, were expressed by expression profile of Bocavirus HBoV1. The HBoV1 profile was transfection in A549 cells, which share the C-terminus with that of the obtained from RNAs following transfections. Analysis of HBoV1 RNA full length NS1. produced during infection quantitatively by Northern or RNase protection assay is currently precluded by the lack of isolated viruses Cellular localization of HBoV proteins at a high load and the low efficiency of virus replication (Dijkman et al., 2009). However, previous analysis of RNAs generated by We next examined the cellular localization of HBoV1 proteins by plasmid transfection of simian parvovirus (Liu et al., 2004), B19V transfection of pHBoV1 in A549 cells. Staining with ant-NS1 C- (Guan et al., 2008) and adeno-associated viruses (Qiu and Pintel, terminus antiserum showed a clear nuclear staining (Fig. 5, NS1), 2002; Qiu et al., 2002) show similar profiles to those seen in virus suggesting that the NS1, NS1*66 and NS1*34 were localized in the infections. Our results showed that the overall transcription profile of nucleus. Furthermore, anti-NP1 staining showed that the NP1 was HBoV1 is similar to that of two other Bocavirus members, MVC and localized in the nucleus as well (Fig. 5, NP1). These results suggest that BPV1 (Sun et al., 2009; Qiu et al., 2007), in that all the species of the NS1 and NP1 of the HBoV1 have their potential function in HBoV HBoV1 mRNAs are transcribed from a single promoter and further DNA replication, similar to those of MVC (Sun et al., 2009). The HBoV1 processed through alternative splicing and alternative polyadenyla- VP1 and VP2, as stained with anti-VP1/2 antiserum, were basically tion. These features are also similar to the transcription profiles of localized in the nucleus with some diffused cytoplasmic staining members in the genera Erythrovirus and Amdovirus (Ozawa et al., (Fig. 5, VP1/2), similar to the capsid proteins of other parvoviruses 1987; Liu et al., 2004; Qiu et al., 2006), which highlights the (Vihinen-Ranta and Parish, 2006). importance of the regulation of alternative polyadenylation in the expression of parvovirus genomes that have a single promoter (Qiu Expression of all HBoV1 proteins does not induce apoptosis or cell cycle and Pintel, 2008). arrest HBoV protein expression The large non-structural protein NS1 of other parvoviruses, e.g. the NS1 of MVM, H-1 and B19V, has been shown to induce apoptosis and/ We observed two major species of HBoV1 mRNAs, the spliced R1M or cell cycle arrest of transfected cells (Brandenburger et al., 1990; and R2M mRNAs, in HBoV1 RNA from transfected cells. These mRNA Daeffler et al., 2003; Ozawa et al., 1988; Rayet et al., 1998; Sol et al., species were not recognized previously (Dijkman et al., 2009). The 1999). The cytopathic and cell cycle regulatory effect elicited during R1M and R2M mRNAs were spliced at the small intron at a level of

Fig. 5. Cellular localization of HBoV1 proteins by transfection. A549 cells were transfected with the pHBoV1 construct. At 48 h posttransfection, cells were stained with anti-NS1, anti- NP1 and anti-VP1/2, respectively. Confocal images were taken at ×60 magniﬁcation (objective lens). Nuclei were stained with DAPI. pBluescript SK(+)-transfected cells were used as control (Control). 152 A.Y. Chen et al. / Virology 403 (2010) 145–154

These multiple small proteins of NS1 might be translated from alternative initiation sites or speciﬁcally cleaved by protease. The exact mechanism by which these small NS1 proteins are produced awaits further investigation.

Effect of HBoV proteins on cells

In parvoviruses, the large nonstructural proteins, NS1 (Nuesch and Rommelaere, 2006; Rayet et al., 1998; Sol et al., 1999) or Rep 78 (Schmidt et al., 2000) are pro-apoptotic proteins. Recently, we have identified the small nonstructural protein 11 kDa of B19V behaves as a potent apoptosis-inducer during infection (Chen et al., 2010b). Parvovirus infection-induced cell death often connects to the pathogeneses of parvovirus infection, e.g., B19V infection-induced cell death of erythroid progenitor cells of bone marrow causes chronic anemia in immunocompromised patients (Young and Brown, 2004). We have recently identified capsid protein expression of AMDV activates caspases (Cheng et al., 2010). Conversely, all the HBoV1 proteins, NS1, NS*66, NS*34, NS1-70, NP1 and VP1/VP2, do not induce apoptosis by transfection (Fig. 6), which is similar to those proteins of Fig. 6. Expressing of HBoV1 proteins does not induce cell cycle arrest or activate MVC, another member in genus Bocavirus (Chen et al., 2010a), and caspases. A549 cells were transfected with the pHBoV1. The transfected cells were maybe a common feature of the Bocavirus. We have recently identified stained with DAPI and anti-NS1 for cell cycle analysis and pan-FLICA and anti-NS1 for detection of apoptosis, respectively, at 48 h posttransfection. Numbers shown in DAPI that replication of the MVC genome activates caspases that accounts staining are percentage of cells at the G0/G1 phase, and numbers shown in FLICA for MVC infection-induced apoptosis (Chen et al., 2010a). However, staining are percentage of pan-FLICA positive cells. A representative result is shown currently an efficient HBoV infection system has not been established; from two independent experiments. whether HBoV1 or HBoV2 infection causes cell death and the role of these proteins in the pathogenesis of HBoV infection await further approximately 60% (Fig. 1C). In addition, a corresponding protein NS1, investigation. which contains a C-terminus of aa 639–781, was detected in HBoV1 transfected cells. This result suggests that the NS1 must be encoded by Materials and methods the R1M and R2M mRNAs, through which the NS1 ORF extends its C- terminus by removing the small intron (Fig. 3). The encoding Cells and transfection sequence of the extended NS1 C-terminus overlaps with the NP1- encoding sequence, but in different frames. Moreover, both MVC and Human alveolar epithelial A549 cells (ATCC CCL-185), human BPV1 encode an NS1, which has the C-terminus-encoding sequences embryonic kidney 293 cells (ATCC CRL-10852) were maintained in overlapped with the NP1-encoding sequence (Qiu et al., 2007; Sun Dulbecco's modified Eagle's medium with 10% fetal calf serum in 5% et al., 2009). BPV1 NS1 protein is expressed at a size of approximately CO2 at 37 °C. 100 kDa (Qiu et al., 2007). The large nonstructural protein NS1 of Transfection was performed using the Lipofetamine™ and Plus™ parvovirus is a multifunctional protein that plays a key role in virus reagent (Invitrogen) or LipoD293 transfection reagent (SignaGen replication. It possesses a site-specific DNA binding domain at the N- Laboratories) following the manufacturers' instructions. terminus, a transcription activation domain at the C-terminus, and enzymatic domains of ATPase and helicase in the middle (Cotmore Plasmid constructs and Tattersall, 2006). The C-terminus of HBoV1 NS1 does not share any similarities with that of the NS1 of other parvoviruses except for HBoV constructs the NS1 of MVC and BPV1. The NS1 C-terminus of the three Bocavirus Incomplete HBoV1 and HBoV2 genomes of 5299 nts were members shares 50% of consensus sequences and 27% identity, amplified from DNA samples extracted from nasopharyngeal aspirate especially the two conserved motifs, at the last 44 amino acids and stool samples, respectively, with primers based on the published (Fig. 3B). Therefore, we believe that the HBoV1 NS1 protein is most HBoV1 sequence (Genbank accession no.: DQ000496) and HBoV2 likely a typical Bocavirus NS1 protein that must have functions, such as sequence (Genbank accession no.: GQ200737), respectively. The transactivation, in virus replication. HBoV1 and HBoV2 DNAs (nt 1–5299) were cloned into XhoI/XbaI- Two other non-structural proteins are expressed by HBoV1, NS1- digested pBluescript SK(+) vector (Strategene), separately, which 70 and NP1. NS1-70 is likely expressed from unspliced R2m mRNA. resulted in pHBoV1 and pHBoV2, respectively. p5TRHBoV1 was made The mid-ORF encoded NP1 was expressed at a high level following by inserting the HBoV1 sequence (nt 1–5299) into an AAV5 ITRs- transfection in comparison with NS1 (Fig. 4A), which is consistent containing plasmid, pAV5ITR (Guan et al., 2008); thus, in p5TRHBoV1, with the abundant level of R3 and R4 mRNAs (Fig. 3A). Unique to all the HBoV1 sequence was flanked with AAV5 ITRs at two ends. bocaviruses so far characterized is the expression of the mid-ORF- pHBoV1(HA-NS1) and pHBoV1(NP1-HA) were constructed by insert- encoded NP1. To our surprise, the NS1 of HBoV1 and HBoV2 are ing an HA tag into nt 259 at the N-terminus of the NS1 ORF and nt expressed in different sizes, resulting in multiple bands on Western 3056 at the C-terminus of the NP1 ORF in pHBoV1, respectively. blot analysis (Fig. 4B). Multiple bands centering at 45 kDa also were pHBoV1(NS1-70-HA) was constructed by inserting the NS1-70 ORF of observed in BPV1 infected cell lysates immunoprecipitated using HBoV1 (nt 253–2169) into BamHI/XhoI-digested pcDNA3CHA. The convalescent sera from BPV1-infected calves (Lederman et al., 1983). pcDNA3CHA vector was constructed by inserting three repeated HA Cleavage of AMDV NS1 has been proved to be essential and functional tag between XhoI and XbaI sites into pcDNA3 (Stratagene). for viral DNA replication (Best et al., 2003). The small bands centralized at 66 kDa were present in both HBoV1 and 2 NS1 Constructs for RNA probe generation expression, suggesting that this small NS1*66 protein must play a HBoV1 transcription units were mapped by HBoV1 probe clones, role during virus infection, which is currently under investigation. created by inserting HBoV1 nt 161–320 (PD1), nt 854–1040 (PA1-2), A.Y. Chen et al. / Virology 403 (2010) 145–154 153 nt 1998–2160 (PA1-2), nt 1998–2223 (PA1-2/D2), nt 2191–2434 The samples were analyzed on a three-laser flow cytometer (LSR II, (PA2/D3), nt 2961–3120 (PA3) and nt 3291–3440 [P(pA)p] sepa- BD Biosciences) within an hour of staining at the Flow Cytometry Core rately into BamHI/HindIII-digested pGEM4Z vector (Promega). These of the University of Kansas Medical Center. Flow cytometry data were probes are depicted in Fig. 1A. analyzed using FACS DIVA software (BD Biosciences).

Constructs for glutathione S-transferase (GST)-fusion expression The HBoV1 NP1 ORF (nt 2410–3067), the C-terminus encoding Acknowledgments sequence (nt 2241–2666) of the HBoV1 NS1, and the C-terminus encoding sequence (nt 4160–4480) of the HBoV1 VP1 were cloned This work was supported by PHS grant RO1 AI070723 from NIAID into pGEX4T3 (GE Health) as pGEX-HBoVNP1, pGEX-HBoVNS1(aa and grant P20 RR016443 from the NCRR COBRE program to JQ, and 640–781) and pGEX-HBoVVP1(aa 369–475), respectively. AI46458 and AI21302 to DJP. All the nucleotide (nt) numbers of HBoV1 and HBoV2 refer to the nucleotide (nt) numbers of the HBoV1 KU2 isolate (Genbank accession no.: GQ925675) and HBoV2 KU1 isolate (Genbank accession References no.: GQ200737). The HBoV1 and HBoV2 genomes were sequenced at the MCLab (www.mclab.com), and all the clones with probes or ORFs Albuquerque, M.C., Rocha, L.N., Benati, F.J., Soares, C.C., Maranhao, A.G., Ramirez, M.L., fi Erdman, D., Santos, N., 2007. Human bocavirus infection in children with were sequenced to ensure their delities. gastroenteritis, Brazil. Emerg. Infect. Dis. 13, 1756–1758. Allander, T., Tammi, M.T., Eriksson, M., Bjerkner, A., Tiveljung-Lindell, A., Andersson, B., RNA isolation, RNase protection assay (RPA) and Northern blot analysis 2005. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc. Natl. Acad. Sci. U. S. A. 102, 12891–12896. Allander, T., Jartti, T., Gupta, S., Niesters, H.G., Lehtinen, P., Osterback, R., Vuorinen, T., p5TRHBoV1 was transfected with pHIVAV5Rep and pHelper Waris, M., Bjerkner, A., Tiveljung-Lindell, A., van den Hoogen, B.G., Hyypia, T., plasmids as previously described into 293 cells (Guan et al., 2008). Ruuskanen, O., 2007. Human bocavirus and acute wheezing in children. Clin. Infect. fl Dis. 44, 904–910. pHBoV1 was transfected into 60% to 80% con uency of A549 cells. Arden, K.E., McErlean, P., Nissen, M.D., Sloots, T.P., Mackay, I.M., 2006. 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